During development of the vertebrate nervous system, neuronal precursors acquire distinct identities based on their position in the early neural tube. For motoneurons, a key component of this identity is the ability to recognize specific guidance cues in the periphery and to project to correct muscle target regions. The long-term objective of this application is to understand how the positional identity of motoneurons is encoded in the early neural tube and subsequently translated into a set of specific axon patterns. The specific aims of this project center on the roles of Hox genes in the specification of motoneuron identity in limb innervating regions of the spinal cord. Hox genes encode transcription factors implicated in the early patterning of multiple structures including the central nervous system and its motoneuron populations. In humans, mutations in Hox genes are associated with limb and genital abnormalities. We have specifically chosen the avian lumbosacral (LS) spinal cord/hind limb as a model system because a substantial background exists on early motoneuron programming and crucial molecular and cellular steps in patterning the limb and spinal cord. Thus, this model provides an opportunity to precisely define the roles of Hox genes in the multistep process of motoneuron patterning. In preliminary studies, Hoxd10 has been ectopically expressed in thoracic segments via in ovo electroporation. Results indicate that the misexpression of this single gene can lead to a posteriorization of the molecular profile and axon trajectories of thoracic motoneurons. In this proposal, preliminary studies will be extended to more fully characterize motoneuron projections and temporal aspects of Hoxd10 function. In ovo electroporation will next be used to mis express Hoxd10 in mesoderm tissues normally encountered by motoneuron axons. Axon tracing will be used to define axon projection and to dissect central from peripheral effects of Hoxd10 expression. Similar approaches will be taken for Hoxa10, a combination of Hoxa10+ Hoxd10, and Hoxd11 to identify unique and cooperative functions of 5'Hox genes. In the above experiments, molecular markers of different types of motoneurons and target regions will be used to assess changes in regional identity following Hox misexpression and to search for potential downstream targets of Hox. Our studies will specifically search for links between Hox proteins and Eph/ephrin receptors and ligands, LIM-Homeodomain proteins, and Meis transcription factors. In addition to chick gain-of-function models, this last aim will take advantage of a loss-of-function model, a Hoxd10 null mouse. Project results will yield fundamental information on motoneuron development and provide insights into the origins of human patterning defects present at birth.